Patents Represented by Attorney Stephen C. Macevicz
  • Patent number: 6265163
    Abstract: The invention provides a method and materials for monitoring and isolating differentially expressed genes. In accordance with the method of the invention, differently labeled populations of DNAs from sources to be compared are competitively hybridized with reference DNA cloned on solid phase supports, e.g. microparticles, to provide a differential expression library which, in the preferred embodiment, may be manipulated by fluorescence-activated cell sorting (FACS). Monitoring the relative signal intensity of the different fluoresent labels on the microparticles permits quantitative analysis of expression levels relative to the reference DNA. Populations of microparticles having relative signal intensities of interest can be isolated by FACS and the attached DNAs identified by sequencing, such as with massively parallel signature sequencing (MPSS), or with conventional DNA sequencing protocols.
    Type: Grant
    Filed: August 6, 1998
    Date of Patent: July 24, 2001
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Glenn Albrecht, Sydney Brenner, Robert B. DuBridge
  • Patent number: 6258569
    Abstract: Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
    Type: Grant
    Filed: November 8, 1999
    Date of Patent: July 10, 2001
    Assignee: The Perkin-Elmer Corporation
    Inventors: Kenneth J. Livak, Susan J. A. Flood, Jeffrey Mamoro, Khairuzzaman Bashar Mullah
  • Patent number: 6235475
    Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.
    Type: Grant
    Filed: August 7, 1998
    Date of Patent: May 22, 2001
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Sydney Brenner, Glenn Albrecht, Stephen C. Macevicz
  • Patent number: 6228589
    Abstract: A method is provided for assessing the toxicity of a compound in a test organism by measuring gene expression profiles of selected tissues. Gene expression profiles are measured by massively parallel signature sequencing of cDNA libraries constructed from mRNA extracted from the selected tissues. Gene expression profiles provide extensive information on the effects of administering a compound to a test organism in both acute toxicity tests and in prolonged and chronic toxicity tests.
    Type: Grant
    Filed: February 28, 2000
    Date of Patent: May 8, 2001
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Brenner
  • Patent number: 6175002
    Abstract: An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5′ strands and the use of adaptors having a 3′ blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3′ blocking groups are removed, the adjacent 5′ hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.
    Type: Grant
    Filed: January 5, 1999
    Date of Patent: January 16, 2001
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Robert B. DuBridge, Glenn Albrecht, Sydney Brenner, Sergei M. Gryaznov, Sarah N. McCurdy
  • Patent number: 6175006
    Abstract: A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.
    Type: Grant
    Filed: March 30, 1999
    Date of Patent: January 16, 2001
    Assignee: The Perkin-Elmer Corporation
    Inventors: William A. Andrus, Christie D. McCollum, Gerald Zon
  • Patent number: 6172218
    Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.
    Type: Grant
    Filed: June 5, 1998
    Date of Patent: January 9, 2001
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Br{umlaut over (e)}nner
  • Patent number: 6172214
    Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.
    Type: Grant
    Filed: August 7, 1998
    Date of Patent: January 9, 2001
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Brenner
  • Patent number: 6150516
    Abstract: The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit.An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports.
    Type: Grant
    Filed: November 20, 1998
    Date of Patent: November 21, 2000
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Sydney Brenner, Glenn Albrecht, Stephen C. Macevicz
  • Patent number: 6140489
    Abstract: The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as "antisense monomers" or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches.
    Type: Grant
    Filed: October 30, 1998
    Date of Patent: October 31, 2000
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Brenner
  • Patent number: 6138077
    Abstract: The invention provides a computerized method, associated apparatus, and computer program product for determining a set of non-hybridizing oligonucleotides. The invention represents a first oligonucleotide in the computer's memory, generates a set of oligonucleotides, including the first oligonucleotide, that meet a specified condition that determines whether the generated oligonucleotides will not hybridize with the first oligonucleotide. The invention also examines each of the generated oligonucleotides in the set to remove oligonucleotides from the set that hybridize with other nucleotides in the set. Thus, the invention develops a minimally cross-hybridizing set of oligonucleotides that can be used for tracking, identifying, and/or sorting classes or subpopulations of molecules by the user of oligonucleotide tags.
    Type: Grant
    Filed: June 3, 1998
    Date of Patent: October 24, 2000
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Brenner
  • Patent number: 6048974
    Abstract: Compounds referred to herein as oligonucleotide clamps are provided that stably bind to target polynucleotides in a sequence-specific manner. The oligonucleotide clamps comprise one or more oligonucleotide moieties capable of specifically binding to a target polynucleotide and one or more pairs of binding moieties covalently linked to the oligonudeotide moieties. In accordance with the invention, upon annealing of the oligonucleotide moieties to the target polynucleotide, the binding moieties of a pair are brought into juxtaposition so that they form a stable covalent or non-covalent inkage or complex. The interaction of the binding moieties of the one or more pairs effectively clamps the specifically annealed oligonucleotide moieties to the target polynucleotide.
    Type: Grant
    Filed: April 30, 1998
    Date of Patent: April 11, 2000
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Sergei M. Gryaznov, David H. Lloyd
  • Patent number: 6013445
    Abstract: The invention provides a method of nucleic acid sequence analysis based on the ligation of one or more sets of encoded adaptors to the terminus of a target polynucleotide. Encoded adaptors whose protruding strands form perfectly matched duplexes with the complementary protruding strands of the target polynucleotide are ligated, and the identity of the nucleotides in the protruding strands is determined by an oligonucleotide tag carried by the encoded adaptor. Such determination, or "decoding" is carried out by specifically hybridizing a labeled tag complement to its corresponding tag on the ligated adaptor.
    Type: Grant
    Filed: October 7, 1997
    Date of Patent: January 11, 2000
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Glenn Albrecht, Sydney Brenner, Robert B. DuBridge, David H. Lloyd, Michael C. Pallas
  • Patent number: 5969119
    Abstract: Disclosed are oligonucleotides probes which are useful for determining a sequence of nucleotides in a target ploynucleotide. In one embodiment, the probe is of the formula: HO-(3')(B)j(5')-OP(.dbd.O-)NH-(B)k-Bt*, wherein B is a nucleotide or an analog thereof; j is in range of from 1 to 12; is in the range of from 0 to 12, such that the sum of j and k is less than or equal to 12; and Bt* is a labeled chain-terminating moiety. In s second embodiment, the probe has a formula selected from the group consisting of (1) OP(.dbd.O)(O--)O--(5.dbd.)(B)sRRRR(B)wBt*, and (2) OP(.dbd.O)(O--)(5')(B)sRRRR(B)w(3')OP(.dbd.O)(O--)O, wherein: is a deoxyribonucleotide or an analog thereof; R is a ribonucleotide; s is in the range of from 1 to 8; w is in the range of from 0 to 8, such that the sum of j and k is less than or equal to 8; and Bt* is a labeled chain-terminating moiety.
    Type: Grant
    Filed: June 10, 1997
    Date of Patent: October 19, 1999
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Stephen C. Macevicz
  • Patent number: 5965720
    Abstract: Modified oligonucleotides 3'-NHP(O)(O.sup.-)O-5' phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.
    Type: Grant
    Filed: January 10, 1997
    Date of Patent: October 12, 1999
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Sergei M. Gryaznov, Ronald G. Schultz, Jer-kang Chen
  • Patent number: 5962228
    Abstract: A novel "primer walking" method for DNA sequencing is provided that comprises repeated cycles nucleotide identification by selective extension and primer advancement along a template by template mutation. An important feature of the invention is providing a set of primers, referred to herein as "rolling primers" that contain complexity-reducing nucleotides for reducing the number of primers required for annealing to every possible primer binding site on a sequencing template. Another important feature of the invention is the systematic replacement of at least one of the four nucleotides in the target polynucleotide with its cognate complexity-reducing nucleotide or complement thereof. Sequencing is initiated by annealing rolling primers differing only in their terminal nucleotides to a primer binding site of a sequencing template so that only the rolling primer whose terminal nucleotide forms a perfect complement with the template leads to the formation of an extension product.
    Type: Grant
    Filed: August 22, 1997
    Date of Patent: October 5, 1999
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Brenner
  • Patent number: 5935527
    Abstract: A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.
    Type: Grant
    Filed: April 29, 1993
    Date of Patent: August 10, 1999
    Assignee: The Perkin-Elmer Corporation
    Inventors: William A. Andrus, Christie D. McCollum, Gerald Zon
  • Patent number: 5888737
    Abstract: An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5' strands and the use of adaptors having a 3' blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3' blocking groups are removed, the adjacent 5' hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.
    Type: Grant
    Filed: April 15, 1997
    Date of Patent: March 30, 1999
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Robert B. DuBridge, Glenn Albrecht, Sydney Brenner, Sergei M. Gryaznov, Sarah N. McCurdy
  • Patent number: 5863722
    Abstract: The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as "antisense monomers" or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches.
    Type: Grant
    Filed: June 7, 1995
    Date of Patent: January 26, 1999
    Assignee: Lynx Therapeutics, Inc.
    Inventor: Sydney Brenner
  • Patent number: 5859233
    Abstract: The invention provides a method of synthesizing oligonucleotide N3'.fwdarw.P5' phosphoramidates using an amine-exchange reaction of phosphoramidites in which a -deprotected 3'-amino group of a solid phase supported oligonucleotide chain is exhanged for the amino portion of a 5'-phosphoramidite of an incoming monomer which has a protected 3'-amino group. The resulting internucleotide phosphoramidite linkage is then oxidized to form a stable protected phosphoramidate linkage. The method of the invention greatly improves product yields and reduces reagent usage over currently available methods for synthesizing the above class of compound.
    Type: Grant
    Filed: December 20, 1996
    Date of Patent: January 12, 1999
    Assignee: Lynx Therapeutics, Inc.
    Inventors: Bernard L. Hirschbein, Karen L. Fearon, Sergei M. Gryaznov, Sarah N. McCurdy, Jeffrey S. Nelson, Ronald G. Schultz